Understanding protein folding is a central life process and is essential for many of the primary quests of structural biology including structure prediction, protein design, mapping evolutionary pathways, learning how mutations cause disease, drug design, and relating structure to function. Consequently, there has been an enormous effort over the years to understand how proteins fold. Essentially all of the effort has been directed to water soluble proteins, however, leaving us largely in the dark about membrane proteins. The gap in our knowledge has occurred, not because membrane proteins are inherently less important, but because they are technically challenging to study. We have been working to understand how membrane protein structures are defined by their amino acid sequence and their complex environment. In prior grant periods we have made membrane protein folding increasingly accessible to detailed examination and have been able to experimentally probe fundamental aspects of membrane protein structure including hydrogen bond strengths, helix kinks and packing efficiency. We propose to continue our examination of key structural features of membrane proteins and to continue developing techniques to advance this still fledgling field. The specific aims are: Aim I. How are kinks induced in transmembrane helices? A hallmark of transmembrane helices is the large fraction of kinks. Thus, an important question in membrane protein folding is how kinks are generated. We will use double mutant cycle analysis to identify residues that collaborate in kink formation. Aim II. How are new strongly polar side chains accommodated in membrane protein tertiary structure? Strongly polar side chains play prominent structural and functional roles in membrane proteins. Thus, the evolutionary process must encounter and tolerate new polar substitutions. Yet mutations to or from polar side chains are the most common disease-causing changes, so there are clearly dangers that must be understood. We therefore propose discover the structural consequences of introducing single Asn residues at different points in the bacteriorhodopsin structure. Aim III. Develop a steric trapping to study the unfolding of membrane proteins under native conditions. The folding of large helical membrane proteins must currently be done in detergents. We will develop a new method for controlling the folding equilibrium within natural bilayers or bilayer-like environments. This development will be a major advance in the field because it will finally allow us to study how the protein and the membrane environment collaborate to fold membrane proteins.